Diagnostic composition for detecting glucose



United States Patent 3,072,539 DIAGNOSTIC COMPOSITION FOR DETECTING GLUCOSE ,OtIs E. Fancher and Dale A. Stautfer, Elkhart, Ind., as-

3,072,539 Patented Jan. 8, 1963 "ice and is therefore easily applicable to carriers, such as filter paper strips, together with the glucose oxidase, the

signm-s to Miles Laboratories Inc" Elkhart, Ind a 5 indicator, and such other ingredients as may be required corporation of India to give the appropriate reaction conditions. No Drawing. Filed Nov. 6, 1959, Ser, N 851,241 The metalloporphyrin used in the embodiments of this 11 Claims. (Cl. 195-1035) invention is a mixture of isomeric urohemins or uroferriporphyrin chlorides (C H N O FeCl), which are char- This invention relates tea new and mproved diagnosacterized by four acetic acid and four propionic acid tic composition and is particularly concerend with a diaggroups attached to the porphyrin ring. The following IIOSUC test Whlch s useful the qualitative detection structural formula characterizes one isomeric form of its and quantitative determination of glucose in biological complex chemical nature: fluids, such as urine, and wherein the reagent composition is incorporated upon a bibulous carrier. 1 We; m s.

The detection of glucose in urine as well as the deter- HOOLMTZ i cnzcnzcoo'fl mination of its concentration therein is of great impor- .i' tance for diabetic patients who must control their diets so as to regulate their sugar intake and who must frehOOCCH-T quently be guided in this regard by a regular check on l urine glucose. But beyond its usefulness in regular urine testing on known diabetics by both patients and physi- C cians, this glucose indicator may also be used efficiently in routine urinalyses in hospitalsand physicians ofiices, V in diabetes detection screening programs, in the difier- Y entiation of glucosuria from other'meliturias, and the HOOCCHZCEIZ! CHZQOOH like. v

B c us early diagnosis d continued control are so As indicated above the catalyst used in the practice of important in diabetes, a urine-sugar test, to be of greatest this invention is a chemically reproducible t p value, must be conveniently rapid, simple enough for any 'P the mode 0f Preparation being as follows! patient to learn with ease, accurate enough to serve the The porphyrins are mad? in accordance With the clinician, and sensitive enough to reflect variations inthe scription given by MacDonald, Chem- Soc; patients condition. Moreover, the reagent composition 4176, 4134, and 8 1. Chem, 34, 1768 (1956). The t b adequately t bk, metallation of the thus-produced porphyrins is carried Procedures for the detection of sugar in urineare well 0111 ac rding to the general procedure of Erdrnan and known in clinical chemistry. One such procedure utilizes COIWiIl, IACS, 69, 750 The metalloporphyrin Benedicts copper reduction test, another employs a selfintelmediatfis are then hydrolysed as Shown y Fischer heating alkaline copper reduction test in tablet form (US. and Drills D Chemie des Pyffols L L Hdlfte, P- Patent No. 2,387,244), still another involves a test which It has been suggested y MacDonald that the reaction depends solely on the action of enzymes (US. Patent No. vProduct, as mentioned above, y be a mixture of 2,848,308). Although these and other procedures h v urohemins in several isomeric forms wherein the arrangegreatly contributed to the art of diagnosing for urine ment o acetic add aHdVPIOPiORiO acid groups yaIiS- glucose, they have not entirely satisfied the above-men- Separation of the'isomefs is not ffiquifed even ationed requirement. ble for the practice of this invention.

We have now found a novel and highly useful glucose- The urohemin mixture has: suflicient activity to catalyze detecting means which represents an important i the oxidation of sensitive indicators such as 2,7-diaminoment and a fresh approach to the problem of determinflllofene Wi t t a d n. of amiHe'd'OIlOTS} ing, glucose in various materials including body fluids, e amine donors, such'as ll-aminobenzothiazole, nicosuch as urine, by a technique whichutilize's a diagnostic tinic acid and pyridine make it possible to decrease "the composition that rivals even the latest and most comq y of urohemin required w sensitive indicators monly used glucose tests in stability and quantitation. and greatly decrease the time, required for oxidation'of Specifically, we have now found that a certain synthetic less sensitive indicators, such as o-tolid e. metalloporphyrin compound is surprisingly and unexpect- It is understood that such additives as suitable proedly capable of acting as catalyst to induce the, oxidation ted-1V6 agents and Wetting agents may incorporated of indicators by hydrogen peroxide formed when glucose therein as illustrated below.- Furthermore, this glucose is aerobically oxidized in the presence of glucose oxidase. test may contain a suitable inert dye to impart tothe The principles-underlying these'reactions are well known composition a uniform background color as well as an and need not be expounded. appropriate butter system to maintain a desired pH range A diagnostic composition according to the present in- '(pH 3 to 7 with a pH of 5.4 being preferred). vention comprises as essential constituents glucose oxi- Although the test device itself may comprise the redase, a synthetic metalloporphyrin and a color-forming agent composition in the form of a tablet, powder, or substance or indicator, such as 2,7-diaminofluorene, o-

other embodiment, we prefer, to insure ease and simtolidine, leucoindophenols, etc. jplicity of test procedure, to affix the reagent composition This invention represents an improvement over previon bibulous base materials or carriers, such as strips or ously described compositions in that the catalyst is a sticks of filter paper, y ing the component in a chemically reproducible, stable compound rather than suitable solvent, impregnating the bibulous strips with a naturally occurring unstable enzyme mixture of a highly the resulting solution and drying the impregnated test variable composition. Moreover, it is surprising and unstrips. As will be obvious from the examples, it is very expected that very small amounts of the catalysts are .easy to prepare the instant glucose indicator due to its sufiicient to produce the desired effect. Embodiments of manifest simplicity. r this invention alsohave advantages overthe hemin-amino i The invention will now be illustrated in greater detail, omplexes disclosed in patent application Serial bio. not limited, by the following examples:

EXAMPLE I Preparation of the lmpregnating Solution Algin (100 mg.) was stirred with 10 ml. of water until a clear solution resulted. Then 10 ml. of a pH 5.4 buffer solution (prepared by dissolving 352.8 g. of sodium citrate dihydrate and 25.6 g. of anhydrous citric acid in water and diluting to 1000 ml.) was added with stirring. A solution of 2 g. of gelatin and 150 mg. of F. D. & C. Red No. 3 (disodium salt of 9-o-carboxylphenyl-6-hydroxy- 2,3,4,7-tetraiodo-3-isoxanthone) in 100 ml. of hot water was prepared and 10 ml. of this solution was added to the algin-buifer solution with stirring. Then a solution of 100 mg. of 2,7-diaminofiuorene dihydrochloride in 10 ml. of hot 50% ethanol was added. A solution of 500 mg. of glucose oxidase (28,700 U./g., dialyzed and lyophilyzed) in ml. of water was added. The mixture was warmed to 40 and a solution of mg. of the synthetic urohemin in 5 ml. of water was added.

Preparation of Diagnostic Strip A bibulous carrier, such as, filter paper out into narrow strips with a water-impervious barrier of ethyl cellulose one-half inch from the tip were dipped into the impregnating solution so that through the process of submersion and capillary attraction the entire one-half inch of the strip up to the barrier was completely impregnated. These strips were dried in a hot air oven at 100 C. for from 30 minutes to 5 hours.

Procedure of Testing In use an impregnated strip made as described above is dipped into the liquid specimen to be tested. When contacted with urine containing glucose, test strips gave positive reactions in about 10 seconds evidenced by various shades of blue color as follows:

With urine containing 1 or 2% glucose an intense blue color developed in 10 seconds, while with urine containing 0.1% glucose a light blue color developed in 10 seconds. With urines containing 0.25% and 0.5% glucose the blue colors which developed in 10 seconds were of intermediate intensities, the urine containing the most glucose being the darkest blue. When dipped in urine containing no glucose, the sticks underwent no color change. A simple color chart based on this phenomenon may be conveniently prepared for use in estimating the glucose content of the urine being tested.

EXAMPLE II The impregnating solution was prepared as in Example I except that the urohemin (10 mg.) together with 20 mg. of 2-aminobenzothiazole was dissolved in 5 ml. of hot 50% aqueous ethanol and this solution was added to the rest of the mixture.

The diagnostic strips were prepared and tested as in Example I. These sticks were slightly more active than those prepared according to Example I.

EXAMPLE III EXAMPLE IV The impregnating solution was prepared by adding 200 mg. of 2-aminobenzothiazole to the solution as prepared according to Example III. The mixture was stirred and warmed to 40 to dissolve the amine. The sticks were prepared and tested as in Example I. The test sticks had the same order of activity as those prepared according to Example I.

In summary, this invention pertains to a diagnostic composition for the detection of glucose in body fluids, and especially in urine, consisting of a bibulous strip or stick which has been impregnated with a composition comprising glucose oxidase, a metalloporphyrin, namely, an isomeric mixture of urohemins and a color-forming substance, such as, 2,7-diaminofiuorene or o-tolidine which is oxidizable by hydrogen peroxide in the presence of the metalloporphyrin.

What is claimed is:

11. A diagnostic composition for detecting glucose which comprises glucose oxidase, a compound selected from the group consisting of a uroferriporphyrin chloride having the structural formula -ca cn coon and compounds having the same ferriporphyrin nucleus dilfering therefrom only in the arrangement of acetic acid and propionic acid groups and a color-forming substance oxidizable by hydrogen peroxide in the presence of said uroferriporphyrin chloride.

2. A diagnostic composition for detecting glucose which comprises glucose oxidase, a compound selected from the group consisting of a uroferriporphyrin chloride having the structural formula and compounds having the same ferriporphyrin nucleus dilfering therefrom only in the arrangement of acetic acid and propionic acid groups, and a color-forming substance taken from the group consisting of 2,7-diaminofluorene and o-tolidine, oxidizable by hydrogen peroxide in the presence of said uroferriporphyrin chloride.

3. A diagnostic composition for detecting glucose which comprises glucose oxidase, a compound selected from the group consisting of a uroferriporphyrin chloride having the structural formula nooccn HOOCCHZ- CH2CH2COOH noocon cn 011 00011 HOOCCH2CH2- cn coon and compounds having the same ferriporphyrin nucleus diflering therefrom only in thearrangement of acetic acid 5 groups and a color-forming substance taken from the and propionic acid groups and a color-forming substance taken from the group consisting of 2,4-diaminofiuorene and o-tolidine, oxidizable by hydrogen peroxide in the presence of said uroferriporphyrin chloride.

4. A diagnostic composition for detecting glucose which comprises glucose oxidase, a compound selected from the group consisting of a uroferriporphyrin chloride having the structural formula HOOCCHg- -CH CH COOH CC Z 2 2 nooccn m cn coon ,HOOCCH2CH2 cn coon and compounds having the same ferriporphyrin nucleus differing therefrom only in the arrangement of acetic acid and'propionic acid groups and 2,7-diaminofiuorrene, oxidizable by hydrogen peroxide in the presence of said uroferriporphyrin chloride.

5 A diagnostic composition for detecting glucose which comprises glucose oxidase, a compound selected from the group consisting of a uroferriporphyrin chloride having the structural formula I CHZCQOH CH COOH HOOCCH CH and compounds having the same ferriporphyrin nucleus differing therefrom only in the arrangement of acetic acid and propionic acid groups and 2,7-diaminofluorene.

6. A glucose-detecting means consisting of a bibulous carrier impregnated with a diagnostic composition comprising glucose oxidase, a compound selected from the group consisting of a uroferriporphyrin chloride having the structural formula nooccn nooccn CH2CH2CO0H nooccn c'af cn coon nooccn cn cn coon and compounds having the same ferriporphyrin nucleus difiering therefrom only in the arrangement of acetic acid and propionic acid groups and 2,7-diaminofluorene oxidizable by hydrogen peroxide in the presence of the uroferriporphyrin chloride.

7. A diagnostic composition for detecting glucose which comprises glucose oxidase, a compound selected from the group consisting of a uroferriporphyrin chloride having the structural formula nooccnjlm-cn cn coon I -CI-l CH COOH FeCl- I CQOH HOOCCH CH CH COOH and compounds having the same ferriporphyrin nucleus difiering therefrom only in the arrangement of acetic acid and propionic acid groups, a complex-forming compound selected from the class consisting of Z-aminobenzothiazole, nicotinic 'acid and pyridine to activate said uroferriporphyrin chloride and a color-forming substance oxidizable by hydrogen peroxide in the presence of the activated uro'feirrinorphyrin chloride. j 1

8. A diagnostic composition for detecting glucose which comprises glucose oxidase, a compound selected from the group consisting of a uroferriporphyrin chloride having the structural formula nooccn j nooccn -CH2CH2COOH nooccn cn C cooH nooccn cn cu coon and compounds having the same ferriporphyrin nucleus differing therefrom only in the arrangement of acetic acid and propionic acid groups, Z-aminobenzothiazole to activate said uroferriporphyrin chloride by forming a complex therewith and 2,7-diaminofiuorene oxidizable by hydrogen peroxide in the presence of the activated uroferriporphyrin chloride.

10. A diagnostic composition for detecting glucose which comprises glucose oxidase, a compound selected from the group consisting of a uroferriporphyrin chloride having the structural formula N nooc CHgl x CHZCHZCOOH I FeCl- I nooccn cn CHZCOOH HOOCCH2CH2: cfi ooon and compounds having the same ferriporphyrin nucleus difiering therefrom only in the arrangement of acetic acid and propionic acid groups, Z-aminobenzothiazole and 2,7-diaminofiuorene.

11. A glucose-detecting means consisting of a bibulous carrier impregnated with a diagnostic composition comprising glucose oxidase, a compound selected from the group consisting of a uroferriporphyrin chloride having the structural formula nooccn HOOCCH CIi and compounds having the same ferriporphyrin nucleus differing therefrom only in the arrangement of acetic acid and propionic acid groups, Z-aminobenzothiazole to activate said uroferriporphy rin chloride by forming a complex therewith, and 2,7-diaminofluorene oxidizable by hydrogen peroxide in the presence of the activated uroferriporphyrin chloride.

References Cited in the file of this patent UNITED STATES PATENTS 2,848,308 Free Aug. 19, 1958 FOREIGN PATENTS 203,451 Australia Sept. 27, 1956 OTHER REFERENCES Chemical Abstracts, vol. 37 (1943), Par. 5423 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3,072,539 January 8, 1963 Otis E. Fancher et a1.

It is hereby certified that error appears in the above numbered patent requiring correction and that the said Letters Patent should read as corrected below.

Column 1,, line 11, for "'concerend" read concerned line 44 for "requirement" read requirements column 3 line 10 for "9-o-carb0xylphenyl-" read 9ocarb0xypheny1- column 5, line 1 strike out "groups and a c0l0r-forming substance taken from the".

Signed and sealed this 21st-day of April 1964.

SEAL Attest:

ERNEST W. *SWIDER EDWARD J. BRENNER Attesting Officer Commissioner of Patents 

11. A GLUCOSE-DETECTING MEANS CONSISTING OF A BIBULOUS CARRIER IMPREGNATED WITH A DIAGNOSTIC COMPOSITION COMPRISING GLUCOSE OXIDASE, A COMPOUND SELECTED FROM THE GROUP CONSISTING OF A UROFERRIPROPHYRIN CHLORIDE HAVING THE STRUCTURAL FORMULA 